Succinate, also called butanedioic acid, is an excellent platform chemical, which is extensively used in the fields of chemical industry, material, medicines, and food industry, and is considered as one of the 12 most valuable platform chemicals by U. S. Department of Energy (McKinlay et al. 2007, Appl Microbiol Biotechnol 76:727-740). Currently, succinate is mainly used in esterification solvent, deicer, engine coolant, food flavour, water treatment chemicals etc. Succinate can also be used for producing many downstream products, such as 1,4-butanediol, tetrahydrofuran, γ-butyrrolactone, N-methylpyrrolidone and 2-pyrrolidone. Besides, succinate and 1,4-butanediol can be polymerized to produce PBS (poly-butylene succinate) plastics, which is a biodegradable plastics with excellent properties. It is estimated that the future market potential of succinate would exceed 2,700,000 tons per year. About 250 chemical products (produced on the basis of benzene material) can all be produced by using succinate as a raw material (McKinlay et al. 2007, Appl Microbiol Biotechnol 76:727-740).
Currently, the production of succinate is mainly based on petrochemical routes using maleic anhydride as raw material. The prices of petroleum greatly fluctuate in recent years, which seriously limit the sustainability and price stability of succinate production. On the other hand, chemical synthesis has complicated processes and usually requires high pressure and high temperature, which greatly increase the energy and material costs during the production; and additionally chemical synthesis also results in serious environmental pollution. The development of high performance bio-manufacturing technology of succinate can fundamentally solve the disadvantages of petrochemical routes, such as ensuring the stable price of succinate without being influenced by the fluctuation of petroleum prices, decreasing the manufacture cost for PBS plastics to facilitate its further applications; realizing green sustainable production, simplifying production process, saving energy and reducing emission, and decreasing environmental pollution. Further, the bio-manufacturing process of succinate can also absorb carbon dioxide, which is a good promotion for low-carbon economics. The core of succinate bio-manufacturing technology is a microbial strain that can effectively convert biomass materials into succinate.
Currently, there are mainly two categories of succinate fermentation bacteria. The first are bacteria that naturally produce succinate, including Actinobacillus succinogens (Guettler et al. 1996, U.S. Pat. No. 5,504,004), Anaerobiospirillum succiniciproducens (Glassner and Datta 1992, U.S. Pat. No. 5,143,834), Mannheimia succiniciproducens (Lee et al. 2002, Appl Microbiol Biotechnol 58:663-668) and Basfia succiniciproducens (Scholten et al. 2009, Biotechnol Lett 31:1947-1951). The other are engineered bacteria that are modified through metabolic engineering, which are mainly E. coli. 
Although natural succinate-producing bacteria can produce succinate in high titers, they have disadvantages. During fermentation, the conversion rate of sugar to succinate is low, and a considerable portion of carbon flux flows into the synthesis of other organic acids. Further, the fermentation of natural succinate-producing bacteria requires rich medium, which increases the production cost as well as the downstream isolation-purification cost, limiting their large-scale industrial production. E. coli only accumulates small amount of succinate during sugar fermentation, but it has a clear physiological and genetic background and is easy to be modified. Many research institutes choose E. coli as starting bacteria, and modify it as engineered bacteria that can produce succinate with high yield.
Phosphoenolpyruvate (PEP) is a key precursor in succinate synthesis pathways. The carboxylation of PEP into oxaloacetic acid (OAA) is a key step in succinate synthesis pathways. Millard et al. increased the yield of succinate by 3.5 times through over-expressing E. coli PEP carboxylase gene ppc (Millard et al., 1996, Appl Environ Microbiol 62: 1808-1810). Kim et al. discovered that overexpression of PEP carboxykinase gene pck in wild-type E. coli showed no influence on succinate production, but overexpression of pck gene in E. coli with ppc gene deletion could increase the yield of succinate by 6.5 times (Kim et al., 2004, Appl Environ Microbiol 70:1238-1241). Kwon et al. of South Korea further discovered that, when the fermentation broth contains bicarbonate ions at high concentration, overexpression of pck gene in wild-type E. coli could increase the yield of succinate by 2.2 times (Kwon et al., 2006, J Microbiol Biotechnol 16:1448-1452).
Chatterjee et al. constructed an engineered strain NZN111 by inactivating pyruvate formate lyase gene pflB and lactate dehydrogenase gene ldhA in E. coli. This strain cannot grow with glucose as carbon source, but can produce succinate, acetate and ethanol by using lactose, fructose, mannose or fructose as carbon source. On this basis, the mutant strain AFP111, which can reuse glucose as carbon source to grow during fermentation, was screened out (Chatterjee et al., 2001, Appl Environ Microbiol 67:148-154; Donnelly et al., 1998, Appl Biochem Biotechnol 70-72:187-198). Vemuri et al. further increased the yield of succinate by over-expressing Rhizobium etli pyruvate carboxylase gene pyc in AFP111. During dual-phase fermentation (first aerobic cultivation, and then anaerobic fermentation to produce acids), the final concentration of succinate could reach 99.2 g/L (841 mM), with a sugar-acid conversion rate of 1.1 g/g (1.68 mol/mol) (Vemuri et al., 2002, J Ind Microbiol Biotechnol 28:325-332).
Sanchez et al. constructed an engineered strain SBS550MG by inactivating alcohol dehydrogenase genes adhE and ldhA, acetate kinase gene ackA, phosphate acetyltransferase gene pta, and isocitrate lyase regulatory protein gene iciR. During dual-phase fermentation (first aerobic culture, and then anaerobic fermentation to produce acids), it could produce 40 g/L (339 mM) of succinate, with a yield of 1.06 g/g (1.61 mol/mol) (Sanchez et al., 2005, Metab Eng 7:229-239).
The recombinant E. coli strains constructed by Vemuri et al. and Sanchez et al. can produce succinate in high titer, but still have disadvantages. The fermentation process employed therein is dual-phase fermentation, i.e. first using an aerobic process to grow the cell culture, and then an anaerobic process to perform fermentation. The operation of such processes is complicated, and the aerobic process greatly increases the cost for device construction and operation. Such recombinant E. coli strains require rich medium, which greatly increases the material cost for fermentation, and results in higher calculated succinate yield.
Jantama et al. constructed a recombinant E. coli strain KJ073 by inactivating ldhA, adhE, formate transporter gene focA, pflB, ackA, methylglyoxal synthetase gene mgsA and pyruvate oxidase gene poxB as well as by subjecting to metabolic evolution. Using mineral salt medium, it can produce 79 g/L (668 mM) of succinate under anaerobic conditions, with a yield of 0.79 g/g (1.2 mol/mol) (Jantama et al., PCT/US2008/057439; Jantama et al., 2008a, Biotechnol Bioeng 99:1140-1153). Recombinant E. coli strain KJ122 was constructed by further inactivating propionate kinase gene tdcD, 2-ketone methyl butyrate lyase/pyruvate formate lyase gene tdcE, aspartate aminotransferase gene aspC and malic enzyme gene sfcA as well as by subjecting to metabolic evolution. Using mineral salt medium, it can produce 80 g/L (680 mM) of succinate under anaerobic conditions, with a yield of 0.89 g/g (1.36 mol/mol) (Jantama et al., PCT/US2008/057439; Jantama et al., 2008b, Biotechnol Bioeng 101:881-893). By metabolic evolution, these two recombinant E. coli strains improved the ability of producing succinate. Zhang et al. constructed a recombinant E. coli strain XZ721 by deleting PEP-phosphosugar transferase I genes ptsI and pflB as well as by enhancing the activity of PEP carboxykinase (PCK). Using mineral salt medium, it can produce 39 g/L (327 mM) of succinate under anaerobic conditions, with a yield of 0.82 g/g (1.25 mol/mol) (Zhang et al., PCT/US2010/029728; Zhang et al., 2009b, Appl Environ Microbiol 75:7807-7813).
In order to increase the titer and/or yield of succinate produced by E. coli, it is desired to further modify metabolic pathways of E. coli. 